Hepatitis B is a major public health problem with more than 350 million persons chronically infected worldwide, 20 to 40% of them being at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite the existence of effective preventive vaccines, the hepatitis B virus (HBV) infection is still rampant in many countries, even developed ones, with an estimation of 4.5 millions of new cases of infection per year worldwide. Unlike the WHO recommendation which is to implement universal vaccination, the coverage of full course preventive vaccination varies from 25% in Asia to 75-90% in Europe. Currently hepatitis B is the 10th cause of mortality (around 1 million of deaths/year) and HBV related liver carcinoma, the 5th most frequent cancer. Geographic repartition of HBV infection is uneven with prevalence lower than 1% in Western countries to more than 10% in South Eastern countries, most part of Africa and Equatorial South America.
Hepatitis B virus is a member of the hepadnaviridae and primarily infects the liver, replicating in hepatocytes. The infectious particles are the so called 42-45 nm “Dane particles” which consist of an outer lipoprotein envelope with three different surface proteins (HBs) and an inner nucleocapsid, the major structural protein of which is the core protein (HBcAg). Within the nucleocapsid is a single copy of the HBV genome linked to the viral polymerase protein (P). In addition to 42-45 nm virions, the blood of HBV-infected patients contains 20-nm spheres made of HBsAg and host-derived lipids which are released from infected cells. These spheres outnumber the virions by a factor of 104-106.
After virions enter hepatocytes, by an as-yet-unknown receptor, nucleocapsids transport the genomic HBV DNA to the nucleus, where the relaxed circular DNA is converted to covalently closed circular DNA (cccDNA). The cccDNA functions as the template for the transcription of four viral RNAs, which are exported to the cytoplasm and used as mRNAs for translation of the HBV proteins. The longest (pre-genomic) RNA also functions as the template for HBV replication, which occurs in nucleocapsids in the cytoplasm. Some of the HBV DNA and polymerase-containing capsids are then transported back to the nucleus, where they release the newly generated relaxed circular DNA to form additional cccDNA. With a half-life longer than the one of hepatocytes, the cccDNA is responsible for the persistence of HBV. Other capsids are enveloped by budding into the endoplasmic reticulum and secreted after passing through the Golgi complex.
Structural and functional organization of the HBV genome has been investigated for more than 30 years. The HBV genome is a relaxed circular partially double-stranded DNA of approximately 3,200 nucleotides consisting of a full-length negative strand and a shorter positive strand. It contains 4 overlapping open reading frames (ORFs), C, S, P and X. The C ORF encodes the core protein (or HBcAg), a 183 amino acid-long protein constitutive of the nucleocapsid and a second protein found in the serum of patients during virus replication known as HBeAg which contains a precore N-terminal extension and a part of HBcAg. The C-terminus of the core protein is very basic and contains 4 Arg-rich domains which are predicted to bind nucleic acids as well as numerous phosphorylation sites. The S ORF encodes three surface proteins all of which have the same C terminus but differ at their N-termini due to the presence of three in-frame ATG start codons that divide the S ORF into three regions, S (226 amino acids), pre-S2 (55 amino acids) and pre-S1 (108 amino acids), respectively. The large-surface antigen protein (L) is produced following translation initiation at the first ATG start codon and comprises 389 amino acid residues (preS1-preS2-S). The middle surface antigen protein (M) results from translation of the S region and the pre-S2 region starting at the second start ATG whereas the small surface antigen protein of 226 amino acids (S, also designated HBsAg) results from translation of the S region initiated at the third start ATG codon. The HBV surface proteins are glycoproteins with carbohydrate side chains (glycans) attached by N-glycosidic linkages. The P ORF encodes the viral polymerase and the X ORF a protein known as the X protein which is thought to be a transcriptional activator.
The viral polymerase is about 832-845 amino acid residues long according to the HBV genotype and it is encoded in a long open reading frame (“P”) that overlaps the 3′ end of the core gene and all the surface protein genes. The viral polymerase is a multifunctional protein composed of four domains, including three functional domains, respectively the terminal protein, polymerase and RNase H domains that catalyse the major steps in HBV replication (priming, DNA synthesis and removal of RNA templates) as well as a non-essential spacer domain present between the terminal protein and polymerase domains (see for example Radziwill et al., 1990, J. Virol. 64:613; Bartenschlager et al., 1990, J. Virol. 64, 5324). The catalytic sites responsible for enzymatic activities have been characterized. In this regard, four residues forming the conserved YMDD motif (residues 538 to 541 numbered with respect to the 832 residue long polymerase) have been shown essential to the DNA- and RNA-dependent DNA polymerase activity whereas RNase H activity is based on a DEDD motif involving four non-consecutive amino acid residues, respectively Asp (D) in position 689, Glu (E) in position 718, Asp (D) in position 737 and Asp (D) in position 777 as well as few other amino acid residues including Val (V) in position 769 and Thr (T) in position 776. Different mutations have been described in the art that abolish the RT polymerase and RNase H activities (Chang et al., 1990, J. Virol. 64: 5553; Bartenschlager et al., 1990, J. Virol. 64, 5324, Radziwill et al., 1990, J. Virol. 64:613 and Chen et al., 1996, J. Virol. 70:6151). Several groups have succeeded in expressing HBV polymerase protein in various host system, but its expression has been reported toxic for the expressing cells, requiring the use of inducible promoters (Choi et al., 2002, Antiviral Res. 55:279; Karimi et al., 2002, J. Virol. 76:8609).
A number of preclinical and clinical studies have emphasized the importance of CD4+ and CD8+ T cell immune responses for effective anti-viral response. It was indeed observed that patients naturally having recovered from hepatitis B mounted multi-specific and sustained responses mediated by T helper (TH) and cytotoxic T (CTL) lymphocytes which are readily detectable in peripheral blood. Appearance of anti-HBe and anti-HBs antibodies indicates a favorable outcome of infection. HBsAg-specific antibodies are neutralizing, mediate protective immunity and persist for life after clinical recovery.
Chronic HBV infection is, however, only rarely resolved by the immune system. The vast majority of chronically infected patients show weak and temporary CD4 and CD8 T cell immune responses that are antigenically restricted and ineffective to clear viral infection. The reason for this alteration of the effector functions of the cellular immune response in chronic hepatitis B is currently not well-understood even if the involvement of different inhibitory molecules that are up-regulated in HBV chronically infected patients, such PD-1, CTLA4 . . . etc, has been observed. Therefore, there is a need for immunomodulatory strategies capable of inducing an effective T-cell response.
Conventional treatment of chronic hepatitis B includes pegylated interferon-alpha (IFNa) and nucleoside/nucleotide analogues (NUCs) such as lamivudine, and more recently entecavir, telbivudine, adefovir and tenofovir. IFNa is a potent antiviral molecule, whereby inhibiting viral replication, which however, causes serious side effects in merely 25-30% of patients. NUCs act as competitive inhibitors of HBV polymerase aimed to inhibit the reverse transcription of the pre-genomic RNA into the negative DNA strand and then the double stranded viral DNA. They limit the formation of new virions, but are ineffective to eliminate the supercoiled cccDNA hidden in the nucleus of infected hepatocytes which constitutes a source of new progeny viruses. This can explain why NUC efficacy is temporary and viral rebound occurs immediately after cessation of treatment, requiring patients to stay lifelong under treatment. In addition, long-term efficacy is also limited due to emergence of resistant HBV mutants (more than 24% after one year and approximately 66% after four years of lamivudine treatment as reported in some studies although newer NUCs showed much fewer occurrences of drug-resistant HBV mutants). A number of HBV strains exhibiting a decreased sensitivity to anti-viral agents have now been isolated and genome sequencing revealed high spot of substitution mutations in the polymerase domain, including in the YMDD motif (US2008-0233557; Zoulim and Locarnini, 2009, Gastroenterology, 137:1593).
Besides antiviral therapies, efforts are currently made to develop supplemental therapies aiming at improved host's immune responses, specifically those mediated by cytotoxic T and helper T lymphocytes. Several encouraging vaccine strategies have focused on HBV surface proteins S, preS1 and/or preS2 (Zanetti et al., 2008, Vaccine 26: 6266; Mancini-Bourguine et al., 2006, Vaccine 24:4482) as well as on multivalent immunotherapy approaches aimed to simultaneously target multiple HBV antigens. For example, immunization with a polyepitope DNA vaccine encoding multiple envelope, core and polymerase epitopes was shown to elicit CTL and TH responses in preclinical mouse models (Depla et al., 2008, J. Virol. 82: 435). An approach based on a mixture of DNA plasmids encoding HBsAg, HBcAg and HBV polymerase (WO2005/056051; WO2008/020656) demonstrated specific anti-HBV cellular and humoral responses in transgenic mouse model of chronic hepatitis B (Chae Young Kim et al., 2008, Exp. Mol. Medicine 40: 669). Phase I clinical trials were initiated in South Korea in HBV carriers in combination with lamivudine treatment (Yang et al., 2006, Gene Ther. 13: 1110). Another approach recently investigated involves the use of a vectored therapeutic vaccine encoding a combination of HBc and HBV polymerase together with Hbs immunogenic domains (WO2011/01565). Mice immunized with Ad-vectorized vaccine showed T cell response against all expressed HBV antigens, especially against polymerase.
One may expect that HBV will continue to be a serious global health threat for many years due to the chronic and persistent nature of the infection, its high prevalence, the continuing transmission of HBV and the significant morbidity of the associated diseases. Thus, there is an important need to develop more effective approaches for improving prevention and treatment of HBV infections or HBV-associated diseases or disorders. In particular, there still exists a need for approaches that conciliate T cell-mediated immunity against the targeted HBV antigen(s), especially against core, and low potential toxicity. Such approaches are especially useful for treating subjects chronically infected with HBV.
This technical problem is solved by the provision of the embodiments as defined in the claims.
Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.